Title: Respiratory supercomplexes act as a platform for complex <scp>III</scp> ‐mediated maturation of human mitochondrial complexes I and <scp>IV</scp>
Abstract: Article8 January 2020Open Access Source DataTransparent process Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV Margherita Protasoni Margherita Protasoni orcid.org/0000-0001-6557-461X Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Rafael Pérez-Pérez Rafael Pérez-Pérez orcid.org/0000-0001-7726-5873 Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain Search for more papers by this author Teresa Lobo-Jarne Teresa Lobo-Jarne orcid.org/0000-0002-1632-438X Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain Search for more papers by this author Michael E Harbour Michael E Harbour Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Shujing Ding Shujing Ding Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Ana Peñas Ana Peñas Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain Search for more papers by this author Francisca Diaz Francisca Diaz Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author Carlos T Moraes Carlos T Moraes orcid.org/0000-0002-8077-7092 Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author Ian M Fearnley Ian M Fearnley Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Massimo Zeviani Massimo Zeviani orcid.org/0000-0002-9067-5508 Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Department of Neurosciences, University of Padova, Padova, Italy Search for more papers by this author Cristina Ugalde Corresponding Author Cristina Ugalde [email protected] orcid.org/0000-0002-9742-1877 Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Madrid, Spain Search for more papers by this author Erika Fernández-Vizarra Corresponding Author Erika Fernández-Vizarra [email protected] orcid.org/0000-0002-2469-142X Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Margherita Protasoni Margherita Protasoni orcid.org/0000-0001-6557-461X Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Rafael Pérez-Pérez Rafael Pérez-Pérez orcid.org/0000-0001-7726-5873 Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain Search for more papers by this author Teresa Lobo-Jarne Teresa Lobo-Jarne orcid.org/0000-0002-1632-438X Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain Search for more papers by this author Michael E Harbour Michael E Harbour Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Shujing Ding Shujing Ding Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Ana Peñas Ana Peñas Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain Search for more papers by this author Francisca Diaz Francisca Diaz Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author Carlos T Moraes Carlos T Moraes orcid.org/0000-0002-8077-7092 Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author Ian M Fearnley Ian M Fearnley Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Massimo Zeviani Massimo Zeviani orcid.org/0000-0002-9067-5508 Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Department of Neurosciences, University of Padova, Padova, Italy Search for more papers by this author Cristina Ugalde Corresponding Author Cristina Ugalde [email protected] orcid.org/0000-0002-9742-1877 Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Madrid, Spain Search for more papers by this author Erika Fernández-Vizarra Corresponding Author Erika Fernández-Vizarra [email protected] orcid.org/0000-0002-2469-142X Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Margherita Protasoni1, Rafael Pérez-Pérez2, Teresa Lobo-Jarne2, Michael E Harbour1, Shujing Ding1, Ana Peñas2, Francisca Diaz3, Carlos T Moraes3, Ian M Fearnley1, Massimo Zeviani1,4, Cristina Ugalde *,2,5 and Erika Fernández-Vizarra *,1 1Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK 2Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain 3Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA 4Department of Neurosciences, University of Padova, Padova, Italy 5Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Madrid, Spain *Corresponding author. Tel: +34 91 7792784; E-mail: [email protected] *Corresponding author. Tel: +44 1223 252700; E-mail: [email protected] The EMBO Journal (2020)39:e102817https://doi.org/10.15252/embj.2019102817 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondrial respiratory chain (MRC) enzymes associate in supercomplexes (SCs) that are structurally interdependent. This may explain why defects in a single component often produce combined enzyme deficiencies in patients. A case in point is the alleged destabilization of complex I in the absence of complex III. To clarify the structural and functional relationships between complexes, we have used comprehensive proteomic, functional, and biogenetical approaches to analyze a MT-CYB-deficient human cell line. We show that the absence of complex III blocks complex I biogenesis by preventing the incorporation of the NADH module rather than decreasing its stability. In addition, complex IV subunits appeared sequestered within complex III subassemblies, leading to defective complex IV assembly as well. Therefore, we propose that complex III is central for MRC maturation and SC formation. Our results challenge the notion that SC biogenesis requires the pre-formation of fully assembled individual complexes. In contrast, they support a cooperative-assembly model in which the main role of complex III in SCs is to provide a structural and functional platform for the completion of overall MRC biogenesis. Synopsis The mitochondrial respiratory chain (MRC), necessary for aerobic cellular energy transduction in eukaryotic cells, consists of five large enzyme complexes that can assemble into larger supramolecular structures called supercomplexes (SCs). Biogenesis of the human MRC requires the cooperative and interdependent action of respiratory SCs. Complex III is a master regulator of MRC maturation and SC formation. Lack of respiratory complex III halts the assembly of complex I by preventing the incorporation of the NADH-module, but it does not induce the degradation of fully assembled complex I. Coenzyme Q and oxidoreductase activity of complex III are required for the maturation of complex I. Mis-assembly of complex III affects the biogenesis of complex IV as it causes the sequestration of unassembled complex IV subunits into complex III preassemblies. Complex I, III and IV assemble in a cooperative way, interacting with each other prior to the formation of the individual complexes. Introduction The mitochondrial respiratory chain (MRC) complex III (cIII) or bc1 complex is a trans-inner-membrane enzyme that couples the transfer of electrons from ubiquinol (reduced coenzyme Q or CoQ) to cytochrome c with the translocation of protons from the mitochondrial matrix to the intermembrane space, by means of the Q-cycle catalytic mechanism (Trumpower, 1990). Biochemically, cIII occupies a central position in the MRC, since it receives electrons from complex I (cI) and complex II (cII) through CoQ and donates them to complex IV (cIV) via cytochrome c. CIII is also at the crossroads of alternative electron transfer pathways, such as those from the glycerol-3-phosphate dehydrogenase, electron transfer flavoprotein (ETF), sulfide–quinone reductase (SQR), and dihydroorotate dehydrogenase (DHODH), all converging onto CoQ (Lenaz et al, 2007). The quaternary structure of the complex is always dimeric (cIII2), with each monomer being composed of ten different subunits (Iwata et al, 1998; Berry et al, 1999, 2000), only one of which (MT-CYB) is encoded by the mitochondrial genome (mtDNA). Structurally, cIII2 is part of all known respiratory supercomplexes (SCs), where it physically interacts with both cI and cIV in the SCs cI+cIII2+cIVn (Schagger & Pfeiffer, 2000), structures known as "respirasomes" because they are in principle able to transfer electrons from NADH to O2 (Acin-Perez et al, 2008; Gu et al, 2016; Letts et al, 2016; Wu et al, 2016; Guo et al, 2017). It is well known that severe cIII2 deficiency in patients carrying null mutations in genes encoding some cIII2 structural components and assembly factors are associated with a concomitant decrease in cI activity (Lamantea et al, 2002; Bruno et al, 2003; Acin-Perez et al, 2004; Barel et al, 2008; Tucker et al, 2013; Carossa et al, 2014; Feichtinger et al, 2017) and, in some cases, in cIV activity as well (Carossa et al, 2014). These pleiotropic effects have been traditionally interpreted as a loss of cI and cIV stability in the absence of their SC partner (Acin-Perez et al, 2004), which is based on the premise that the biogenesis of MRC SCs proceeds by the incorporation of pre-made fully assembled individual complexes (Acin-Perez et al, 2008). Here, we have used proteomics and biogenetic approaches to comprehensively characterize the biogenesis and organization of the MRC components in a homoplasmic MT-CYB null mutant human cell line, devoid of fully assembled cIII2 (de Coo et al, 1999; Rana et al, 2000; Perez-Perez et al, 2016). Contrary to the current model (Acin-Perez et al, 2004), our data demonstrate that the severe cI deficiency associated with the absence of cIII2 does not originate from destabilization of the fully assembled cI holoenzyme but rather from assembly stalling of nascent cI. MT-CYB mutant cybrid mitochondria accumulate a cI assembly intermediate lacking the catalytic N-module (Mimaki et al, 2012; Moreno-Lastres et al, 2012; Sanchez-Caballero et al, 2016), which is stabilized by the cI assembly factor NDUFAF2 (Ogilvie et al, 2005). In addition, we found that specific cIII2 subunits were recruited into stalled protein structures that sequestered cIV subunits, affecting the maturation of the cIV holo-enzyme. These data explain the molecular mechanisms leading to combined respiratory chain deficiency associated with the absence of cIII2, challenge the concept of SC assembly by incorporation of fully assembled individual complexes, and demonstrate the essential role of SCs as cIII2-driven factories carrying out efficient assembly and maturation of the overall mitochondrial respiratory chain. Results Combined mitochondrial respiratory chain deficiency in Δ4-CYB cybrids Enzymatic activities of respiratory chain complexes I, II, III, and IV were measured in the #17.3E Δ4-CYB clone, homoplasmic for the 4-bp deletion in MT-CYB (heretofore referred to as Δ4-CYB), in comparison with clone #4.1, containing 100% wild-type (heretofore referred to as WT) mitochondrial DNA (mtDNA). Both cybrid clones, obtained from 143B TK− ρ° cells (King & Attadi, 1996a; King & Attardi, 1996b), were populated with mitochondria from the same heteroplasmic patient (Rana et al, 2000). In the Δ4-CYB cells, cIII2 activity was virtually absent and the activities of cI and cIV were significantly lower than the WT values, to 25 ± 13% and 64 ± 11%, respectively (Fig 1A). The profound reduction in cI amounts and activity of Δ4-CYB was confirmed by in-gel activity assays (IGA) and by immunodetection, with a specific antibody against the cI subunit NDUFS1, following blue-native gel electrophoresis (BNGE) separation of the native MRC complexes in mitochondrial extracts from the Δ4-CYB and WT cell lines (Fig 1B and C). The amounts of assembled complexes V and II were not drastically affected by the MT-CYB mutation in two different Δ4-CYB clones: #17.3E (E) and #17.3B (B) (Fig 1C). Figure 1. Complex I and IV enzymatic deficiencies in Δ4-CYB cells The activities (mUnits/g of protein) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and Δ4-CYB cells and normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n = 4–6 biological replicates). Unpaired Student's t-test **P = 0.0100; ***P = 0.0002; ****P < 0.0001. Complex I in-gel activity assays (IGA) after blue-native gel electrophoresis (BNGE) of WT and Δ4-CYB samples solubilized with either 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 h (lighter signals in DDM gels) or were left to continue the reaction for 24 h to obtain darker signals (DDM and Digitonin gels). BNGE, Western blot, and immunodetection, with anti-NDUFS1 (cI), anti-ATP5A (cV), and anti-SDHB (cII) antibodies, of samples from the WT cybrids and from Δ4-CYB clones E (#17.3E) and B (#17.3B). Clone E was the cell line of choice for the analysis shown in panels (A and B), and all the figures hereafter. Source data are available online for this figure. Source Data for Figure 1 [embj2019102817-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint To investigate the origin of the combined respiratory chain deficiency, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, to compare the relative abundance of proteins from mitochondrial extracts of the two cell lines. Both the Δ4-CYB and the WT cybrids were cultured in "Heavy (H)" and "Light (L)" media and then mixed before mitochondrial isolation by cell disruption, differential centrifugation (Fernández-Vizarra et al, 2010), and solubilization with 1.6 mg n-dodecyl β-D-maltoside (DDM)/mg protein. This experiment was performed in duplicate using reciprocal labeling of the mutant and control cells (Vidoni et al, 2017). The resulting fractions were resolved by blue-native gel electrophoresis (BNGE); each lane was then excised in 64 1-mm-thick slices and analyzed by mass spectrometry (MS). This analysis included the relative quantification of 1,263 proteins, the most downregulated of which were structural subunits of cIII and cI (Figs 2A and EV1). The amounts of nine known cI assembly factors (ACAD9, ECSIT, FOXRED1, NDUFAF1, NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF6, and TMEM126B) did not differ significantly between the Δ4-CYB and WT cells (Fig 2A). Conversely, the tenth cI assembly factor detected in this analysis, NDUFAF2 (Ogilvie et al, 2005), and the cII assembly factor SDHAF2 (Hao et al, 2009) were significantly more abundant in Δ4-CYB mitochondria. Other proteins were also upregulated in the mutant cells. These included CHCHD2, whose knock-down causes cIV deficiency (Baughman et al, 2009; Imai et al, 2019), HIGD2A, one of the human orthologs of yeast Rcf1, which stabilizes the interaction between cIII2 and cIV (Chen et al, 2012; Strogolova et al, 2012; Vukotic et al, 2012; Rieger et al, 2017), and GHITM or growth hormone-inducible transmembrane protein, a member of the BAX inhibitor motif-containing (TMBIM) family. GHITM localizes to the inner mitochondrial membrane, interacts with CHCHD2, and is deemed to participate in the maintenance of mitochondrial morphology and integrity (Oka et al, 2008; Meng et al, 2017). Figure 2. Reduced steady-state levels of structural MRC subunits in Δ4-CYB cells Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy-to-light (H/L) and light-to-heavy (L/H) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and Δ4-CYB cells (see also Fig EV1). The logarithmic ratios were calculated using MaxQuant (Cox & Mann, 2008), and the statistical significance of the differences for the enrichment or depletion of the proteins was determined with Perseus (Cox & Mann, 2011; Tyanova et al, 2016). Labeling of the thirteen mtDNA-encoded MRC structural subunits. Cells were incubated with [35S]-L-Met for 1 h in the presence of emetine 100 μg/ml to inhibit cytoplasmic translation. Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two-way ANOVA with Sidak's multiple comparisons test ****P < 0.0001; ***P = 0.0007. Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two-way ANOVA with Sidak's multiple comparisons test ****P < 0.0001; **P = 0.0024 (NDUFS3); **P = 0.0061 (NDUFB8). Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two-way ANOVA with Sidak's multiple comparisons test **P = 0.0011. Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two-way ANOVA with Sidak's multiple comparisons test ****P < 0.0001. Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and Δ4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). There were no differences in the steady-state levels of the tested subunits (2-way ANOVA with Sidak's multiple comparisons test). Source data are available online for this figure. Source Data for Figure 2 [embj2019102817-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Complexome profiling of cIII2-containing structures in samples from in WT and Δ4-CYB cells solubilized with DDM (related to Figs 2, 3, 4) Heatmaps of cIII2 structural subunits and assembly factors derived from the DDM-solubilized samples in the experiment where Δ4-CYB cells were labeled with the heavy (H) amino acids. Black = 0; yellow = 0.5; red = 1 relative peptide intensities of the most frequent peptide found in each of the samples individually. Complexome profiles of the cIII2 structural subunits found in both cell lines in the two reciprocal labeling experiments. The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0 versus the molecular mass, calculated using the individual complexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and Δ4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments. Heatmaps of cIII2 structural subunits and assembly factors derived from the digitonin-solubilized samples in the experiment where WT cells were labeled with the heavy (H) amino acids. Black = 0; yellow = 0.5; red = 1 relative peptide intensities of the most frequent peptide found in each of the samples individually. Profiles of the subunits only found in the WT samples in the two reciprocal labeling experiments. The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1 versus the molecular mass, calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The represented values are the mean ± SEM of the two reciprocal labeling experiments. Download figure Download PowerPoint To further examine the expression of the structural components of the MRC complexes and the ATP synthase (cV) in the Δ4-CYB cells, we performed metabolic labeling of the thirteen mtDNA-encoded subunits. This analysis indicated that only MT-CYB was not translated in the Δ4-CYB cybrids and that there was no clear reduction in the synthesis of any of the seven ND (cI) subunits (Fig 2B). Next, we tested the steady-state levels of several cI, cII, cIII2, cIV, and cV subunits from whole-cell lysates, separated by SDS–PAGE, and immunovisualized by Western blot (WB) with specific antibodies. CIII2 subunits were in general the most reduced in the Δ4-CYB cybrids, with significantly lower levels of UQCRC1, UQCRC2, UQCRB, UQCRFS1, and UQCRQ (Fig 2C). Conversely, cytochrome c1 (CYC1) showed comparable steady-state levels in both cybrid lines. Likewise, immunodetection using an anti-UQCRQ monoclonal antibody (Abcam ab110255) visualized a band of molecular mass smaller than 10 kDa and revealed equal levels in both cell lines (Fig 2C). Subsequent experiments suggested that this antibody fails to reliably detect UQCRQ, whereas it seems to cross react with UQCR10 (Fig 4C). Several cI subunits also showed variable reduction in their steady-state levels, depending on the structural module with which they associate (Stroud et al, 2016; Zhu et al, 2016). The lowest levels were detected for NDUFS1, a component of the catalytic N-module, followed by NDUFB8, belonging to the ND5-module, and NDUFS3, belonging to the Q-module. The amounts of NDUFA9 and NDUFB11, assigned to the ND2 and ND4 modules, respectively, were not significantly affected in the mutant cells (Fig 2D). The steady-state levels of mitochondrial and nuclear-encoded cIV subunits were similar to WT cells, except for significantly lower amounts of COX6B1 (Fig 2E), one of the subunits incorporated in the late stages of cIV assembly (Vidoni et al, 2017). SDHB of cII was also markedly reduced, to about half in Δ4-CYB cybrids compared with WT (Fig 2F), whereas cV subunits showed no differences between the two cell lines (Fig 2G). The absence of MT-CYB causes accumulation of specific CIII assembly intermediates Differentially labeled mitochondria solubilized with either 4 mg digitonin (Figs 3 and EV1A and B) or 1.6 mg DDM/mg protein were resolved by BNGE (Fig EV1C and D). Each lane was excised in 64, 1-mm-thick slices, and "complexome profiles" of protein distribution through the gel were obtained by LC/MS analysis (Heide et al, 2012; Vidoni et al, 2017). By this approach, we compared the relative migration and abundance of the cIII2 components in Δ4-CYB versus WT clones, in conditions that allow the analysis of individual MRC complexes (DDM) and supercomplexes (SCs, visualized in digitonin-solubilized samples). As expected, fully assembled cIII2 and cIII2-containing SCs were missing in the Δ4-CYB cells (Fig EV1). Peptides corresponding to six structural cIII2 subunits, UQCRC1, MT-CYB, UQCRH, UQCRB, UQCRQ, and UQCR11 and to one assembly factor, UQCC2, were found only in the datasets corresponding to WT mitochondria (Fig EV1), reflecting again their virtual absence in Δ4-CYB cells. UQCC2 binds to nascent MT-CYB in the very early stages of cIII2 assembly (Gruschke et al, 2012; Tucker et al, 2013), and its presence seems to depend on the existence of a functional MT-CYB. In contrast, peptides corresponding to the remaining four subunits, UQCRC2, CYC1, UQCRFS1, and UQCR10; and two assembly factors, BCS1L and MZM1L (LYRM7), were detected in both control and mutated mitochondria (Figs 3A and D, and EV1). To validate the findings obtained from the complexome profiling, we performed BNGE followed by WB and immunodetection analyses of digitonin-solubilized mitochondria. 2D BNGE of Δ4-CYB samples (Fig 3B) confirmed the presence of CYC1 in several protein structures ranging from low to high molecular mass, and the absence of UQCRC2, UQCRFS1, and UQCRQ from cIII2 structures and from higher molecular size bands visible in Δ4-CYB. Conversely, these subunits were always found in the cIII2 holocomplex and canonical SCs of WT cells (Fig 3B). Indeed, in the Δ4-CYB cells, residual amounts of UQCRC2, CYC1, and UQCRQ subunits were immunodetected in a high-molecular size area of 2D-BNGE WB, which does not correspond to that of WT SCs and does not contain UQCRFS1. This protein aggregate was also detected in the top gel slices by MS complexome analyses (Fig EV2). Figure 3. Blue-native gel electrophoresis (BNGE) mass spectrometry and immunodetection analysis of cIII2-related proteins Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised (see also Figs EV1 and EV2). The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, versus the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and Δ4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments. Second-dimension BNGE of digitonin-solubilized samples from WT and Δ4-CYB cells, Western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles. Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII2 subunits. The x-axis values were the slice number (1-64), and the y-axis values were the relative peptide intensity. The graph shows the mean ± SD (n = 2). Two-way ANOVA with Sidak's multiple comparisons test **P = 0.0083 (UQCRC2); **P = 0.0033 (UQCRFS1); *P = 0.0224; n.s. = non-significant. Complexome profiles of two cIII2 assembly factors (BCS1L and LYRM7 or MZM1L) generated in the same way as in (A). The represented values are the mean ± SEM of the two reciprocal labeling experiments. Source data are available online for this figure. Source Data for Figure 3 [embj2019102817-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Complexome profiling and area quantification of internal control proteins (related to Fig 3)The complexome profiles of the three chosen proteins, citrate synthase (CS), and two members of the translocase of the outer membrane family (TOM20 and TOM22) were generated as in the main Fig 3. The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1 versus the molecular mass, calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines were determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and Δ4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated proteins. The x-axis values were the slice number (1–64), and the y-axis values were the relative peptide intensity. The plotted values are mean ± SD (n = 2). T